Method for producing dendritic cells

ABSTRACT

Disclosed are embryonic stem cell-derived dendritic cells, genetically modified immature dendritic cells capable of maturation, as well as methods for the production of such cells. In one embodiment, the cells made be produced by a method comprising the steps of providing a population of embryonic stem cells; culturing the embryonic stem cells in the presence of a cytokine or combination of cytokines which brings about differentiation of the embryonic stem cells into dendritic cells; and recovering the dendritic cells from the culture. In a further embodiment, the cells may be genetically modified.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/789,669, filed Apr. 24, 2007 now U.S. Pat. No. 7,473,556, which is acontinuation of application Ser. No. 09/849,499, filed May 4, 2001, nowU.S. Pat. No. 7,247,480, which is a continuation of International PatentApplication No. PCT/GB99/03653, filed Nov. 5, 1999, and claims priorityfrom GB Patent Application Number 9824306.6, filed Nov. 5, 1998. Theentire content of the prior applications is incorporated herein byreference.

The invention relates to a method for the production of dendritic cellsfrom embryonic stem cells and to the dendritic cells so produced. Theinvention also relates to genetically modified embryonic stem cells andtheir use in the production of genetically modified dendritic cells; tomethods for investigating dendritic cells; and to methods forinvestigating the function of mammalian genes.

BACKGROUND OF THE INVENTION The Role of Dendritic Cells in the ImmuneResponse

Dendritic cells (DC) constitute a trace population of leukocytes,originating from the bone marrow but distributed widely throughout mostorgans of the body, with the possible exception of the brain [Steinman1991; Banchereau & Steinman, 1998]. The function of DC is largelydependent on their state of maturation, which varies according to theirlocal microenvironment. DC resident within interstitial tissues, such asthe Langerhans cells of the skin, are predominately immature, forming anetwork of cells adapted to the acquisition of foreign antigensfollowing a local microbial challenge.

To perform such a sentinel function, immature DC are competentphagocytes, taking up whole microorganisms and apoptotic cells forprocessing [Albert et al. 1998a], as well as soluble protein antigens bythe endocytic route. Such activity betrays the close lineagerelationship between DC and macrophages; indeed the classical DC firstdescribed by Steinman and colleagues [1973] are now known to be derivedfrom myeloid progenitors, in common with members of thereticuloendothelial system. What distinguishes DC from macrophages,however, is the nature of their response to an encounter with antigen ata primary site of infection. Inflammatory stimuli, such as the localrelease of interferon-γ or lipopolysaccharide, induce the maturation ofDC precursors [De Smedt et al., 1996; Cella et al., 1997], causing themto lose the ability to acquire further antigens but inducing theirmigration via the draining lymphatics, to the secondary lymphoid organs[Austyn & Larsen, 1990]. Here they adopt a stimulatory role, presentingthe cargo of antigens they acquired in situ, to the repertoire of naiveT cells. Their ability to activate T cells that have never beforeencountered antigen, is a property unique to DC and is a function of theco-stimutatory molecules they express upon maturation, of which CD40,ICAM-1 (CD54), B7-1 (CD80) and B7-2 (CD86) are the best characterized.Furthermore, their propensity to induce a Th1 phenotype among the Tcells which respond is due largely to the secretion of cytokines such asIL-12 and IL-18 [Cella et al., 1996; Koch et al., 1996].

Because of their unrivalled ability to stimulate naive T cells in vivo,all immune responses, whether protective or pathogenic, are initiatedupon the recognition of antigen presented by DC. Consequently, thepotential for modulating the outcome of an immune response by harnessingthe function of DC has aroused widespread interest. Indeed, theirpotential has been successfully exploited in a number of laboratoriesfor enhancing an otherwise inadequate immune response to tumour-specificantigens, resulting in efficient tumour regression [Mayordomo et al.,1995; Celluzzi et al., 1996]. Furthermore, by providing immature DC witha source of chlamydial antigens, Su and colleagues have been able tosuccessfully immunize mice against subsequent infection with Chiamydia[Su et al., 1998], illustrating their likely usefulness in programs ofvaccination against infectious agents that have proven difficult toeradicate using conventional strategies.

Over the past few years, the study of immunology has been revolutionizedby the discovery that DC may present antigen not only for the purpose ofenhancing cell-mediated immunity, but also for the induction ofself-tolerance [Finkelmann et al., 1996; Thomson et al., 1996]. Thiscontention has been supported by the characterization of a secondlineage of DC derived from a lymphoid progenitor in common with T cells[Wu et al., 1997; Shortman & Caux, 1997]. These cells share with‘myeloid DC’ the capacity to acquire, process and present antigen to Tcells but appear to induce unresponsiveness among the cells with whichthey interact, either by preventing their expansion through limitingIL-2 release [Kronin et al., 1996], or provoking their premature deathby apoptosis [Suss & Shortman, 1996]. In this respect, lymphoid DC havebeen reported to constitutively express Fas-ligand which induces celldeath among cells expressing its counter-receptor, Fas. These findingshave raised the additional prospect of further harnessing the propertiesof DC to down-modulate detrimental immune responses, such as thoseinvolved in autoimmune disease and the rejection of allografted tissues.

In spite of the promise DC hold for exploitation in a therapeuticsetting, a number of less-desirable properties of DC have consistentlylimited progress. Firstly, although it is the immunogenic andtolerogenic function of mature DC which is most amenable to immuneintervention, DC exhibit a short life span once terminallydifferentiated. This has made the prospect of genetic modification of DCless attractive since any benefits gained are necessarily short-lived.Furthermore, primary DC are peculiarly resistant to transfection,confounding most attempts to stably express heterologous genes; indeedthe best protocol currently available involves the use of mRNA insteadof cDNA for transfection purposes, creating, at best, a transientexpression system [Boczkowski et al., 1996]. Although many groups haveattempted to circumvent some of these difficulties by generating stableDC lines, the results have been universally disappointing, most putativelines being either retrovirally transformed [Paglia et al., 1993;Girolomoni et al., 1995; Volkmann et al., 1996] or incapable ofprogressing beyond an immature state [Xu et al., 1995]. Thus none ofthese provides a useful, renewable source of DC or one that can begenetically manipulated.

Embryonic Stem Cells and their Differentiation

Embryonic stem (ES) cells are derived from the epiblast of advancedblastocysts. The epiblast cells contribute to all cell types of thedeveloping embryo, rather than the extra-embryonic tissues. IndividualES cells share this totipotency but may be maintained and propagated inan undifferentiated state by culturing them in recombinant leukaemiainhibitory factor (rLIF) [Smith et al. 1988], or on a monolayer ofembryonic fibroblasts which may act as a potent source of this orrelated cytokines. Although ES cells may be propagated for a fewpassages in LIF, for long term culture, fibroblast feeder cells arepreferred since ES cells maintained indefinitely in rLIF may lose theirdifferentiation potential.

Unlike primary cultures of DC, ES cells are particularly amenable togenetic modification since they survive even the most harsh conditionsfor the introduction of foreign DNA, including electroporation.Consequently, ES cells have been used extensively over recent years forthe production of transgenic mice and for gene targeting by homologousrecombination. Indeed, by introducing a null mutation into selectedgenes, it has proven possible to generate ‘knockout’ mice, congenitallydeficient in expression of specific molecules [Fung-Leung & Mak, 1992;Koller & Smithies, 1992].

The ability of ES cells to contribute to all lineages of the developingmouse, once reintroduced into recipient blastocysts, is a property whichhas also proven useful in vitro for the study of lineage relationships[Snodgrass et al., 1992; Keller 1995]. Indeed, a variety of protocolshas been devised to encourage differentiation of ES cells along specificpathways. To date, there have been reports of the emergence of celltypes as diverse as cardiac muscle, endothelial cells, tooth and neurons[Fraichard et al., 1995; Li et al., 1998]. In addition, differentiatingES cells have been shown to engage in the development of haematopoieticstern cells [Palacios et al., 1995] with the potential to differentiateinto erythrocytes, macrophages, mast cells [Wiles & Keller, 1991; Wiles,1993] and lymphocyte precursors of both the T and B cell lineages[Gutierrez-Ramos & Palacios, 1992; Nisitani et al. 1994; Potocnik etal., 1997].

THE INVENTION

It has now been discovered that DC can be generated by culturing EScells under certain conditions, more specifically in the presence ofIL-3 and optionally GM-CSF. Despite the many studies of haematopoiesisfollowing ES cell differentiation in vitro, the appearance of primary DC(i.e. DC not passaged in culture in their own right) has not previouslybeen reported. Surprisingly, while IL-3 has been used in a number ofstudies, either alone or in combination with GM-CSF, to inducehaematopoiesis within developing embryoid bodies [Wiles & Keller, 1991;Keller, 1995] no DC development has been reported, although a cleareffect on erythropoiesis and the development of macrophages and mastcells was routinely observed.

The new findings provide a novel approach to genetic modification of DCwhich makes use of ES cell differentiation in vitro. In particular,stable lines of genetically modified ES cells can be used to generatemutant DC on demand.

Thus, according to a first aspect of the invention there is provided anes dentritic cell (esDC).

As used herein, the term “es” as applied to dentritic cells (DC) isintended to define dentritic cells which are derived from embryonic stem(ES) cells. Thus, esDC cells may be generated directly from ES cells byculture in vitro (for example, as described herein).

In another aspect, the invention provides a genetically modifiedimmature dentritic cell capable of maturation.

The cells of the invention are preferably human cells. Recent reports ofthe derivation of human ES cells [Thomson et al. 1998], have stimulatedmuch interest in their exploitation for the generation ofterminally-differentiated cell types for use in cell replacement therapy[Gearhart 1998; Keller and Snodgrass, 1999]. For many cell types,however, such as neurons, muscle fibres and oligodendrocytes, theireffectiveness in vivo depends on the efficiency with which they can betargeted to the correct anatomical location and site of the originallesion, as well as their propensity to integrate into the host tissueand maintain their physiological competence. For this reason the EStechnology now available is far more likely to find an application amongpopulations of cells such as DC that, once reintroduced in vivo, havebeen shown to migrate under the influence of chemokines, along compexmigratory pathways to secondary lymphoid tissues. Importantly, theskilled worker will readily be able to adapt the protocols describedherein for the generation of DC from human ES cells, for the reasonsexplained below.

Firstly, Thomson and colleagues [1998] made use of embryonic fibroblastsfrom the mouse as a source of feeder cells and found compatibilitybetween the two species, allowing human ES cells to be maintainedlong-term in an undifferentiated state. Secondly, much is now knownabout the growth factors required for the differentiation of mature DCin vitro from human haematopoietic stem cells (HSC) [reviewed inShortman and Caux, 1997]. Significantly, of all the combinations ofcytokines tested, only GM-CSF and IL-3 have been found to have thecapacity to support DC development from CD34+ HSC, although the efficacyof this protocol is greatly enhanced by the addition of TNF-a to theculture medium, suggesting that this cytokine may also facilitate esDCdevelopment from embryoid bodies. Importantly, recombinant humancytokines including GM-CSF, IL-3 and TNF-a are currently available froma number of commercial sources, making the technology readilyaccessible.

Another approach contemplated by the invention achieves germinecompetence by harnessing nuclear transfer technology [Wilmut et al.1997; Wakayama et al. 1998] to permit the transfer of nuclei from humancells to enucleated ES cells of another species (such as ESF116) inorder to confer on the nucleus the propensity for germine transmission.Moreover, nuclear transfer in this way may represent a possible solutionto the complex ethical concerns surrounding derivation of novel human EScell lines, making them more widely-available for purposes such as thegeneration of DC for therapeutic applications.

The invention also provides various medical uses of the cells of theinvention, including therapy and prophylaxis. Particularly preferred areimmunotherapeutic uses.

The invention therefore provides in another aspect a method forproducing dendritic cells which method comprises:

-   -   i) providing a population of embryonic stem cells;    -   ii) culturing the embryonic stem cells in the presence of a        cytokine or combination of cytokines which bring about        differentiation of the embryonic stem cells into dendritic        cells; and    -   iii) recovering the dendritic cells from the culture.

A cytokine which has been found to be of critical importance in thegeneration of DC from ES cells in vitro is IL-3. In the presence of IL-3alone DC develop which exhibit the characteristics of lymphoid ratherthan myeloid DC.

On the other hand, in the presence of a combination of IL-3 and GM-CSF,larger populations of DC appear which represent DC of myeloid origin.

Thus, the invention is concerned with the production of lymphoid-typeand myeloid-type DC under different conditions.

The invention is also concerned with ES cells which are geneticallymodified and which can pass on the genetic modification or modificationsto the resulting DC. Thus, the method according to the invention mayemploy genetically modified ES cells.

The invention also provides dendritic cells produced by the methodsdescribed herein, and genetically modified ES cells useful in themethods described herein including ES cells in which a gene normallyexpressed in dendritic cells is inactivated, and ES cells transfectedwith a construct comprising a promoter which is preferentially active indendritic cells.

In another aspect, the invention provides a method for investigating amammalian gene, which method comprises generating a test population ofdendritic cells from a population of embryonic stem cells and comparingthe test dendritic cells in respect of the gene.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Phase-contrast micrographs of ES cell-derived dendritic cells.(a) Low power view of an embryoid body 24 hr after plating onto tissueculture plastic, showing the emigration of stomal cells in a radialfashion. (b-c) esDC developing around the periphery of a colony. Notethe sharpe demarcation between stromal cells supporting theirdevelopment and those that fail to do so. (d) Appearance of clusters ofesDC (arrows) similar to those apparent in cultures of mouse bonemarrow. (e) esDC that have seeded areas of the dish uncolonized bystromal cells. (f) Cultures of putative lymphoid esDC maintained in IL-3alone.

FIG. 2: shows electron micrographs of esDC cultured in GM-CSF and IL-3;Electron micrographs of esDC cultured in GM-CSF and IL-3 showing typicalDC morphology (a) and a propensity to phagocytose apoptotic cells(arrow), consistent with their immature phenotype. The bar represents 5μm.

FIG. 3: shows surface phenotype of esDC grown is GM-CSF and IL-3assessed by flow cytometry; Surface phenotype of esDC grown is GM-CSFand IL-3 assessed by flow cytometry. Filled histograms indicate levelsof expression of CD44 (a), B7-1 (b), ICAM-1 (c) B7-2 (d), CD40 (e),CD11c (f) and class II MHC (g). Open histograms represent levels ofbackground staining determined using irrelevant species- andisotype-matched control antibodies.

FIG. 4: shows immunostimulatory activity of esDC in the allogeneic mixedleukocyte reaction; Immunostimulatory activity of esDC in the allogeneicMLR. esDC from the CBA/Ca ES cell line ESF116 were co-cultured withpurified T cells form C57B1/10 mice and the extent of proliferation wasmeasured as a function of ³H-TdR uptake 5 days later.

FIG. 5: shows IL-2 secretion by the T cells in response to antigenpresentation by esDC, and inhibition of IL-2 by a mAb to MHC class II;(a) IL-2 secretion by the T cell hybridoma, 2G7.1, in response to HELpresented by live esDC (closed symbols) but not DC that had been fixedfirst in paraformaldehyde to prevent antigen uptake (open symbols). (b)Stimulation of the 2G7.1 hybridoma is inhibited by the addition of a mAbspecific for class II MHC (closed symbols) but not by the addition of anirrelevant species and isotype-matched control antibody (open symbols).

FIG. 6: shows flow cytometric analysis of esDC following maturationinduced by the addition of LPS to cultures; Flow cytometric analysis ofesDC following maturation induced by the addition of LPS to cultures.Filled histograms indicated the levels of expression of class II MHC(a), CD11c (b), B7-1 (c), B7-2 (d), CD40 (e) and ICAM-1 (f). Openhistograms indicate the levels of background staining obtained usingirrelevant species and isotype-matched control antibodies.

FIG. 7: shows immunostimulatory activity of LPS-treated esDC.Immunostimulatory activity of LPS-treated esDC. Mature esDC stimulatethe strong proliferation of naive, allogeneic T cells (closed circles)but only weak proliferation of syngeneic cells (open triangles). At thesame time point, equivalent numbers of immature esDC fail to stimulateeither allogeneic or syngeneic cells (open circles and closed trianglesrespectively).

FIG. 8: shows Immunostimulatory activity by myeloid and lymphoid esDC; Acomparison of the immunostimulatory activity of myeloid (closed circles)and ‘lymphoid’ esDC (open circles).

FIG. 9: shows antigen-processing activity of myeloid and lymphoid esDC.A comparison of the antigen-processing activity of myeloid and lymphoidesDC. At the top dose of DC, the lymphoid population (hatched bar) areconsiderably less able to present antigen to the hybridoma than myeloidDC (filled bar), although both induce widespread cell death.

FIG. 10: shows the generation of esDC stably transfected with GFPfollowing introduction of the transgene Into the parent ES cell line.Generation of esDC stably transfected with GFP following introduction ofthe transgene Into the parent ES cell line. (a) Colony of ESF116 viewedunder fluorescent confocal microscopy showing expression of GFP far inexcess of the level of autofluorescence associated with the monolayer ofembryonic fibroblasts (b). (c)-(d) Embryoid bodies derived from theESF116.EGFP clone showing retention of the transgene duringdifferentiation. (e)-(f) Representative esDC developing from transfectedembryoid bodies viewed under phase contrast (e) and fluorescencemicroscopy (f) confirming expression of GFP by terminally-differentiatedcells.

The source of IL-3 and GM-CSF for use in the invention is not critical;either or both may be provided for example in pure recombinant form, orsecreted from a cell line transfected with the gene and expressing therecombinant protein. In the latter case, tissue culture supernatant fromthe cell line may be used.

So far as concentration is concerned, in the presence of murine IL-3alone murine DC will develop in concentrations as low as 40 U/ml,although 5,000 U/ml is optimal. In practice a concentration of about1,000 U/ml may be preferable since it is economically more viable andthere is still good colony growth of DC at that concentration.

For ES cells in the presence of IL-3 together with GM-CSF, some synergybetween the two cytokines may occur. The cell surface receptors for IL-3and GM-CSF have a common β-chain and therefore quite possibly share someof the same cell signalling mechanisms.

An optimum level of murine GM-CSF for development of murine DC is about30±5 ng/ml. At that level there is receptor saturation. However, GM-CSFat a concentration as low as 0.1 ng/ml stimulates the production oftrace numbers of DC in the presence of 1,000 U/ml IL-3.

Important for the development of DC from ES cells is the formation ofembryoid bodies, which are preferably in liquid suspension culturerather than in any semi-solid matrix. It is preferable that embryoidbodies are free-floating for differentiation to proceed optimally.

Embryoid bodies are formed from ES cells which have been removed fromthe inhibitory effects of LIF. The cells proliferate to form clusters ofviable cells, each of which represents an embryoid body and can comprisedifferentiated or partially differentiated cells of a variety of celltypes.

In a particular embodiment of the method according to the invention,embryoid bodies are plated onto tissue culture dishes and exposed to theappropriate cytokine or combination of cytokines to promote developmentof DC. The embryoid bodies adhere to the surface and give rise tocolonies of stromal cells which migrate outwards. After a few days DCdevelop around the periphery, presumably from early haematopoietic stemcells present in the embryoid bodies. DC which develop in this way canbe harvested in substantially pure form, normally with less than 10%contaminating cell types e.g. about 5 to 10% contaminating cell types.

Prior to the formation of embryoid bodies, the ES cells are routinelymaintained in an undifferentiated state in the presence of LIF. The LIFis generally provided at this stage in pure recombinant form. However,for maintenance of ES cells in long term culture prior to the formationof embryoid bodies, LIF is preferably provided by culturing the ES cellsin the presence of fibroblast feeder cells which secrete LIF and othercytokines.

ES cells for production of DC in the method according to the inventionmay conceivably be derived from any appropriate mammalian source.Illustrated herein are murine ES cells and DC, but it will be clear thatthe invention is not necessarily limited to murine cells. ES cells fromcertain mouse strains are found to be permissive for DC development,while ES cells from other strains are not. However, it will also beclear that the invention is not limited to those permissive strainsdisclosed herein since it is a straightforward matter to prepare EScells from other strains and test them for their competence indifferentiating into DC.

The apparent inconsistency between the results presented herein andprevious studies using ES cells in which no DC were produced orrecovered, may reflect a variety of possible factors. These includedifferences in the protocols employed, an inability in previous studiesto identify any resulting DC, and strain differences in the propensityof ES cells to support DC development. In support of the latterpossibility, initial studies on the CBA/Ca cell line ESF116 wererepeated using a second CBA/Ca line generated in-house (ESF99) and onefrom 129/Sv mice which is widely used for gene knockout technology andwhich is commercially available (D3). Interestingly, while ESF99supported the development of esDC, albeit to a lesser extent thanESF116, D3 failed entirely to do so under the same culture conditions.ES cells generated from other strains can easily be tested for theirability to support development of DC by using the protocols describedherein. An additional example of a mouse strain from which ES cells havebeen shown to support development of DC is C57B1/6 (ESF75).

Certain applications of the invention are discussed in more detail belowand in the Examples which follow. It will be clear that the invention isnot limited to the specific embodiments described herein. In particular,the genetic manipulation of the ES cells may be in any manner whichresults in any useful DC phenotype.

Uses of the present invention extend to the fields of tumourimmunotherapy and vaccination against infectious agents. Examplesinclude transfection of the parent ES cells with genes encodingtumour-specific antigens or candidate microbial antigens against which aprotective immune response is desirable. The endogenous expression ofwhole protein antigens in this way may harness the potent antigenprocessing capacity of DC to select the most appropriate epitopes forpresentation on both class I and class II MHC, effectively by-passingthe need for laborious identification of the epitopes involved.Furthermore, co-transfection of such cells with genes encoding FLIP(accession number: U97076) or bcl-2 (accession number: M16506) mayprolong the life-span of esDC administered in vivo. Both molecules havebeen shown to exert a protective effect, actively interfering with theapoptotic pathways which normally limit DC survival, but in a mannerthat does not induce their transformation [Hockenbery et al. 1990]. Byhaving their lifespan prolonged in this way, esDC presenting foreign ortumour-specific antigens may provide a chronic stimulus to the immunesystem. As an additional advantage, the need for adjuvants for themounting of a powerful protective immune response may be reduced orremoved.

The potential for generating lymphoid DC, thought to be important in themaintenance of peripheral self-tolerance, may be exploited in thetreatment of autoimmune disease which is characterized by loss of thetolerant state. Certain animal models for autoimmune disease will beuseful in investigating the possibilities for treatment. Recently,Goulet and co-workers [1997] reported the isolation of ES cells from theMRL mouse strain susceptible to autoimmunity and demonstrated theirgermline competence. Such cells may prove useful for the production ofesDC of the correct genetic background to permit the development ofstrategies for immune intervention. Alternatively or additionally, EScells established from the diabetes-prone NOD mouse could provide usefulDC for assessing the potential for immune intervention. A successfullyproduced ES cell line could be transfected with GAD-65 (accessionnumber: L16980), an autoantigen known to be involved in the aetiology ofinsulin-dependent diabetes mellitus (IDDM), and induced to differentiatealong the lymphoid route. Upon administration in vivo, such cells mayactively seek out and tolerize T cells specific for the autoantigen,thereby limiting the extent and progression of tissue damage.Furthermore, by introducing the whole gene encoding GAD-65, allpotential epitopes will be presented to the T-cell repertoire,overcoming problems associated with intramolecular determinant spreading[Lehmann et al. 1993]. A similar procedure could be carried out fortolerizing to other autoantigens.

Recently, protocols have been published for the generation of ES cellsin which both alleles of a gene have been targeted by homologousrecombination, resulting in cells deficient in a given protein [Hakem etal., 1998]. This provides an approach for altering DC function byknocking out candidate genes such as the p40 subunit of IL-12 (accessionnumber: M86671) or the p35 subunit of IL-12 (IL-12 is a hederodimer andat least two genes are involved in its expression). Since this cytokineis fundamental to the establishment of a Th1 response, responding Tcells may default to a Th2 phenotype in its absence. Given that Th1 andTh2 cells are mutually antagonistic and that the latter are frequentlyprotective in inflammatory autoimmune conditions [Liblau et a/1995],IL-12⁺ esDC may prove effective in inducing immune deviation andmodulating the outcome of an ongoing autoimmune response. Should theselection criteria for production of knockout ES cells according to thepublished protocols prove to be too stringent, alternative approaches toprevent expression or activity of target molecules can be employed. Suchapproaches include for example antisense constructs, ribozymes or theexpression of dominant negative forms of molecules, where available. Adominant negative form of a molecule is an altered e.g. mutated formwhich blocks the function of the endogenous form of the molecule, forexample by binding in its place. Examples of all of these approaches arepresent in the literature.

Identification of Novel Targets for Immune Intervention

The approaches to immune intervention, outlined above, require priorknowledge of specific genes involved in the immune response and thefunction they perform. Nevertheless, only a small proportion of thegenes that control DC function have been elucidated. The protocols forthe development of DC from ES cells in vitro as described herein may,therefore, be exploited for the identification of novel targets forimmune modulation which may ultimately prove useful in a clinicalsetting.

Several approaches to identifying new genes have recently beendescribed, of which the serial analysis of gene expression (SAGE) isperhaps the most powerful [Valculescu et al., 1995]. This methodologypermits those genes that are actively expressed by two populations ofcells to be compared in a differential manner. It may, therefore, bepossible to compare gene expression in embryoid bodies from ESF116,known to support DC development, and those from D3 which fails to do so.Such an approach may define genes involved in the early stages ofhaematopoiesis which control development of the DC lineage.Alternatively, purified populations of myeloid and lymphoid DC may becompared to elucidate the genes responsible for converting animmunostimulatory DC to one capable of inducing self-tolerance.

While such an approach may highlight important new genes involved in theontogeny and function of DC, there remains a significant ‘gene-functiongap’, it being considerably easier to identify genes that contribute toa particular phenotype than to elucidate the function of the proteinsthey encode. As a way of addressing this deficiency, a number oflaboratories have pioneered gene-trapping technology [Evans et al.,1997] which seeks to trap genes in an unbiased way and provide thepotential for identifying their function. To this end, Zambrowicz et al.[1998] have generated an ‘Omnibank’ of ES cells in which genes have beenrandomly targeted for inactivation. Using these cells, knockout mice maybe generated which may be screened for specific defects which mightbetray the function of the targeted gene. Although the production ofknockout mice is now well-established, the screening of large numbers ofgenes in this way remains an immense undertaking which is likely to belimited by the many logistical constraints. By combining gene trappingtechnology with our own approach and established readouts for antigenprocessing and immunostimulation, we may be able to screen rapidly manynew genes to identify those that confer on DC their unique properties.This strategy may prove attractive to commercial organizations seekingto identify novel targets for the delivery of DC-specific drugs, so asto intervene in the very genesis of the immune response.

EXAMPLES Example 1 Derivation and Maintenance of ES Cells from CBA/CaMice

Although ES cells may be readily generated from 129/Sv and C57B1/6strains of mice, strains used more widely in immunological research,such as CBA/Ca and the diabetes-prone NOD mouse, have proven peculiarlyresistant. Nevertheless, we have recently developed and publishedmethods for their derivation from such strains [Brook & Gardner, 1997],one of which, ESF116, has been used extensively in these studies and wasdeposited with the Belgium Coordinated Collections of Microorganisms(BCCM™), Universiteit Gent, Laboratorium voor MoleculaireBiologie—Plasmid Collection, K. L. Ledeganckstraat 35, B-9000 Gent,Belgium on Jul. 29, 1998 under Accession number LMBP 1668CB, under theterms of the Budapest Treaty. ESF116 is karyotypically male, formschimeras upon injection into recipient blastocysts and has been found totransmit through the germline.

The ESF116 cell line was isolated from an inbred CBA/Ca female mousewhich had been ovariectomized bilaterally and given 1 mg of Depo-Provera(Upjohn, UK) on the afternoon of the third day after mating with a matemouse of the same strain. Blastocysts arrested in development prior toimplantation were recovered in standard HEPES-buffered medium on themorning of the 7th day post-ovariectomy. Each blastocyst was opened upwith a pair of solid-tipped, siliconized glass microneedles mounted onLeitz micromanipulator units, which were used to tear open the muraltrophectoderm. Once opened, the blastocysts were incubated for 24 min at4° C. in Ca²⁺- and Mg²⁺-free phosphate-buffered saline (PBS) containingtrypsin at 5 mg/ml (Difco) and pancreatin (Difco) at 25 mg/ml.Blastocysts were subsequently returned to the micromanipulation chamberand the trophectoderm of each was opened out with the needles so thatthe outer surface of the tissue was spread against the coverslip of thechamber. Using one needle to hold the trophectoderm, the other waslowered from the trophectoderm and moved sideways against the exposedinner cell mass (ICM) so as to dislodge the superficial endoderm fromthe deep epiblast. Once the endoderm had been removed, the same needlewas then raised until it made contact with the trophectoderm upon whichit was moved sideways, so as to scrape the epiblast gently from theoverlying trophectoderm [Gardner, 1985]. Both the initial opening of theblastocysts and their dissection was performed with the microscope stagecooled to 5° C. Once isolated, the epiblast tissue from each blastocystwas pipetted into the individual wells of a four-well tissue cultureplate (Nunclon), that had been seeded with mitotically-inactivatedprimary embryonic fibroblasts the previous afternoon and irrigated withfresh ES cell medium shortly before the epiblasts were explanted.

After culturing for 6 days, individual colonies were picked from thedish using a pulled pasteur pipette, dissociated in a drop oftrypsin-EDTA and replated into a fresh well of feeder cells. Theresulting colonies were cultured for a further 3 days before thecontents were passaged into a 35 mm dish containingmitotically-inactivated embryonic fibroblasts and labelled passage 1.The cells were further expanded into a 25 cm²⁺ tissue culture flaskafter 2 days, passaged into two fresh flasks 2 days later and thereafterpassaged consistently every 3 days.

For routine maintenance of the ESF116 cell line, a stock of embryonicfibroblast feeder cells was prepared from C57B1/6 embryos excised at day12-13 of gestation. Embryonic tissues, with the exception of the headand liver, were finely minced with a sterile surgical blade in PBSsupplemented with 2.5% trypsin (Gibco) and 0.02% EDTA. Aftertransferring to a universal tube, the suspension was placed in awaterbath at 37° C. for 5 min and further dissociated by vigorousshaking. After standing for 5 min, undissociated tissues were found tosediment while unwanted lipids and fats rose to the air-liquidinterface. The cell suspension between these two layers was harvestedand transferred to a tube containing complete medium (DMEM supplementedwith 10% FCS, 2 mM L-glutamine and 5×10⁻⁵ M 2-mercaptoethanol) to haltthe action of the trypsin. Three serial extractions were performed usingtrypsin-EDTA and the resulting cell suspensions pooled and pelleted in abench-top centrifuge. The pellet was resuspended in complete medium anddistributed equally among four 75 cm² tissue culture flasks. Afterreaching confluency, the monolayers of fibroblasts were passaged intofour 150 cm² flasks to permit their expansion. Once confluent, thefibroblasts were harvested, resuspened in complete medium containing 10%DMSO, aliquoted into cryotubes and stored frozen under liquid nitrogenuntil required.

Before use as feeder cells for routine maintenance of ESF116, embryonicfibroblasts were mitotically inactivated by culturing for 2 hr in mediumcontaining 10 μg/ml of mitomycin C (Sigma). The cells were washed inPBS, harvested using trypsin-EDTA and distributed among 25 cm² tissueculture flasks. The fibroblasts were incubated for at least two hours toallow them to adhere to the plastic and form a confluent monolayer.ESF116 cells were seeded onto the awaiting feeder layers by forming asingle cell suspension using trypsin-EDTA and producing a range ofdilutions in ‘ES medium’ consisting of DMEM supplemented with 15% FCS, 1mM sodium pyruvate, 2 mM L-glutamine and 5×10⁻⁵ M 2-mercaptoethanol.

Example 2 Generation of DC from ESF116

Prior to differentiation of ESF116 in vitro, the cells were culturedfree from fibroblast feeder cells by passaging twice in succession in12.5 cm² flasks precoated with 0.1% gelatin in PBS to promote theiradherence. Cells were maintained in an undifferentiated state duringthis period by the addition of 1000 U/ml of rLIF to the culture medium.Since, under these conditions, the ESF116 cells expanded rapidly but thecontaminating fibroblasts remained mitotically inert, the latter wereeventually lost by serial dilution. Pure populations of ESF116 weresubsequently harvested by trypsinization, washed and plated onto 90 mmdishes of bacteriological plastic (Sarstedt) at a density of 3-5×10⁵cells per dish in 20 ml of complete medium lacking rLIF Under theseconditions, the ESF116 cells failed to adhere to the bacteriologicalplastic but remained in suspension where they continued to proliferate,forming embryoid bodies. These spheres continued to increase in size,becoming macroscopic at approximately day 4 of culture and adopting acystic appearance by day 10-12. In the absence of exogenous LIF, theembryoid body has been shown to provide an ideal microenvironment inwhich differentiation proceeds in a manner and with the kineticsexpected of the early mouse embryo [Keller et al. 1993; Schmitt et al.1991].

After 14 days in culture, the embryoid bodies were transferred to a 50ml test tube and allowed to sediment under unit gravity before beingwashed twice in fresh medium to remove unwanted debris. The embryoidbodies were plated at low density onto 90 mm tissue culturedishes(Corning) in 20 ml of ES medium further supplemented with 1000U/ml of recombinant murine IL-3 (R&D Systems) and 2% (v/v) tissueculture supernatant from the X63 cell line transfected with the murinegene encoding GM-CSF The final concentration of GM-CSF was judged to beapproximately 25 ng/ml in bone marrow proliferation assays in which astandard dose-response curve had been constructed using recombinantGM-CSF.

After overnight culture at 37° C., a proportion of the embrycid bodieswas found to adhere to the plastic and give rise to colonies of stromalcells, emigrating outwards in a radial fashion (FIG. 1 a). Byapproximately day 4 of culture, cells bearing distinctive DC morphologyconsistently appeared around the periphery of the stromal layer, thearea of DC growth being sharply demarcated (FIG. 1 b-c). In thisperipheral location, DC continued to proliferate and accumulate,eventually forming the large clusters (FIG. 1 d) characteristic of DCgenerated from bone marrow precursors. With time, DC were found to seedthose areas of the dish still free of underlying stroma, where theirdendritic morphology was particularly apparent (FIG. 1 e). Undertransmission EM, these cells showed the ultrastructural features ofearly DC (FIG. 2 a); indeed the detection of cells which had apparentlyphagocytosed apoptotic cells from their local environment (FIG. 2 b)provides further circumstantial evidence of their identity as immatureDC [Albert et al. 1998b].

ES cell-derived DC (esDC) could be harvested by gentle pipetting andisolated from unwanted debris by passage of the cell suspension over a70 μm cell filter (Falcon). Harvesting of esDC in this manner leftintact much of the stromal layer which supported the growth ofsuccessive cohorts of DC: indeed, cultures have been maintained in ourlaboratory for at least 5 weeks, during which the cells were routinelyharvested for use in experiments. This protocol therefore gives rise tolong-term cultures of DC, overcoming many of the difficultiesencountered when isolating DC from primary tissues.

The requirement for GM-CSF in the generation of esDC is in accordancewith universal findings of its involvement in the development of DCbelonging to the myeloid lineage. What is surprising, however, is theinability of this cytokine to support DC development from ES cells inisolation, although GM-CSF alone is highly effective in the generationof large numbers of DC from bone marrow [Inaba et al, 1992].Furthermore, the need for IL-3 in DC ontogeny is unexpected, althoughnot entirely without precedent. A trace population of CD4 positive Tcells known as “plasmacytoid T cells” which have remained enigmatic formany years, have recently been shown to develop the characteristics ofDC in the presence of interleukin-3 (IL-3) [Grouard et al. 1997]. IL-3has been shown to sustain an early population of haematopoieticprogenitor cells [Sonada et al. 1988], upon which granulocyte macrophagecolony stimulating factor (GM-CSF) may act to induce differentiationalong the DC pathway: in the absence of this progenitor, GM-CSF appearsineffectual.

Example 3 Generation of DC from ESFI 16 Without GM-CSF

Whereas IL-3 is indispensable for the generation of esDC, therequirement for GM-CSF does not appear to be absolute. Embryoid bodiesgenerated as described in Example 2 and plated onto tissue cultureplastic in complete medium containing 1000 U/ml of IL-3 alone, supporteda trace population of cells, in the usual peripheral location, bearingdendritic morphology [FIG. 1 f]. These cells failed to accumulate to thenumbers apparent in cultures described in Example 2 supplemented withGM-CSF and appeared with delayed kinetics after plating. These cellshave a limited functional potential in conventional assays of DCactivity (see below). Together with their ability to develop in theabsence of GM-CSF, this suggests that these cells represent lymphoidrather than myeloid DC, since the lymphoid lineage is known to beindependent of GM-CSF [Saunders et al. 1996]. Thus, we have succeeded indefining the conditions for growth of primary DC of myeloid origin andprimary DC which exhibit characteristics of lymphoid DC, from ES cellsin vitro.

Example 4 Characterization of ES Cell-Derived Dendritic Cells

1. Phenotype

Although irregular morphology is a universal characteristic of DC, anovel source of cells cannot be unequivocally assigned to the DC lineagewithout recourse to determining their surface phenotype and functionalpotential. We have, therefore, prepared esDC as described and analysedtheir expression of surface markers by flow cytometry. Using a panel ofmonoclonal antibodies, we have shown esDC to express high levels of CD44(FIG. 3 a), indicative of their myeloid origin, and the presence of lowbut reproducible levels of the co-stimulatory molecules B7-1 (FIG. 3 b)and ICAM-1 (FIG. 3 c). Although neither B7-2 nor CD40 could be detectedat the cell surface (FIG. 3 d-e), analysis of mRNA by RT-PCR confirmedthe presence of species specific for both molecules, as well as theDC-associated cytokines IL-12 and IL-18 (data not shown). Surprisingly,esDC lacked surface expression of both the DC-specific marker, CD11c(FIG. 3 f), and MHC class II determinants (I-E^(K)) (FIG. 3 g), aphenotype suggestive of their immature status.

2. Activity

Given the potential for expression of many co-stimulatory molecules byesDC, we investigated their ability to stimulate a primary T cellresponse, a function which distinguishes DC from all other APC.Accordingly, incubation of esDC with purified naive T cells fromallogeneic C57B1/10 mice stimulated their proliferation in adose-dependent fashion (FIG. 4), albeit with kinetics delayed by twodays relative to mixed leukocyte reactions (MLRs) involving conventionalsources of DC, such as the spleen and bone marrow. These findings wereagain suggestive of the immaturity of esDC which appeared to requireseveral days for the acquisition of an immunostimulatory phenotype.

Since immature DC may be distinguished from mature cells by theirgreater propensity for antigen processing and presentation, we nextinvestigated the ability of esDC to process the classical foreignantigen, hen eggwhite lysozyme (HEL), for presentation to the T cellhybridoma, 2G7.1, known to be specific for the 1-18 peptide of HEL inthe context of I-E^(K) [Adorini et al., 1993]. Incubation of esDC with2G7.1 induced their activation in an antigen-dependent fashion, leadingto the active release of IL-2 (FIG. 5 a). This activation was inhibited,either by the prior fixation of esDC with paraformaldehyde to preventantigen up-take (FIG. 5 a), or by the addition of a monoclonal antibodyspecific for I-E^(K) (clone 17-3-3S) (FIG. 5 b). These results suggestthat, although immature at the time of harvesting, esDC are induced tomature and express class II MHC determinants upon interaction with Tcells, similar requirements for T cell contact having been reportedpreviously for the maturation of a DC line [Volkmann et al., 1996].

3. Maturation of esDC

Although engagement with T cells in a cognate fashion is the most potentstimulus currently known for DC maturation, studies from variouslaboratories have reported the additional role played by inflammatorymediators such as tumour necrosis factor a (TNF-a) andlipopolysaccharide (LPS) in the acquisition of an immunostimulatoryphenotype. We investigated, therefore, whether the addition of LPS tocultures of esDC would act as a surrogate stimulus for their maturationin vitro, permitting the production of purified populations ofimmunostimulatory esDC that might be exploited for the purpose ofvaccination.

Accordingly, esDC were harvested and replated onto fresh tissue cultureplastic in the presence of 1 μg/ml of LPS (Sigma). After overnightculture, many esDC had acquired a highly irregular morphology with largeveils of cytoplasm and dendrites, characteristic of mature DC. Flowcytometric analysis revealed this population to have stronglyup-regulated class II MHC (FIG. 6 a) and the co-stimulatory moleculesB7-1 (FIG. 6 c), B7-2 (FIG. 6 d), CD40 (FIG. 6 e) and ICAM-1 (FIG. 6 f).Furthermore, these DC expressed low but reproducible levels of CD11c(FIG. 6 b), consistent with their acquisition of a mature phenotype.

When LPS-treated esDC were used as the stimulators of an MLR, they werefound to induce the proliferation of allogeneic, but not syngeneic, Tcells with enhanced kinetics (FIG. 7), the response peaking 48 hoursearlier than was evident in cultures employing immature esDC asstimulators. Furthermore, responses significantly above background wereobtained with as few as 600 esDC per well (FIG. 7), suggesting that, ona cell-by-cell basis, mature esDC are several orders of magnitude morepotent in their capacity to induce primary T cell responses than theirimmature counterparts.

4. Lymphoid DC

We have investigated the functional phenotype of the putative lymphoidDC cultured in IL-3 alone. These cells are less capable of stimulatingnaive T cells in an MLR compared with esDC grown in a combination ofGM-CSF and IL-3 (FIG. 8). Furthermore, although they retain residualcapacity to process and present HEL to the 2G7.1 hybridoma (FIG. 9),they induce widespread apoptotic cell death of the responding T cells,far more than might be expected from the low level of activationindicated by IL-2 release. These data are consistent with theindications that DC cultured in IL-3 alone are closely allied to thelymphoid lineage, actively restricting T-cell responsiveness byinitiating Fas-induced apoptosis.

Example 5 Genetic Modification of esDC—Transfection of ESF116 with GreenFluorescent Protein (GFP)

The ability to generate long-term cultures of primary DC from ES cellsin vitro, provides unparalleled opportunities for the geneticmodification of DC for use in immunotherapy.

ESF116 was transfected with an expression vector containing GFP and astable clone expressing high levels of the transgene was selected forthe generation of esDC. The production of green fluorescent esDC willenable their migration patterns to be studied in vivo. The mammalianexpression vector pEGFP-N1, used in these experiments, is commerciallyavailable (Clontech Catalogue Number: 6085-1; Genbank Accession Number:U55762) and contains a multiple cloning site designed to facilitate theproduction of fusion proteins between heterologous proteins and EGFP atthe N-terminus. EGFP or fusion constructs are expressed under thecontrol of the immediate-early promoter of human cytomegalovirus (CMV).The vector contains a neomycin resistance gene which provides G418resistance for selection of stably transfected mammalian cells.

ESF116 were cultured away from fibroblast feeder cells using recombinantLIF and were harvested as a single cell suspension which was adjusted to2×10⁶ cells/ml. 800 μl of cell suspension were mixed with 40 μl ofpEGFP-N1 at 1 μg/ml which had previously been linearized by incubationwith the restriction endonuclease ApaL1 (BioLabs) for 3 hrs at 37° C.ESF116 were electroplated using 450V, a capacitance of 25 μF. and a timeconstant of 0.5 sec and were placed on ice for 10 min. The cells werethen distributed equally into two 25 cm² tissue culture flaskscontaining monolayers of embryonic fibroblasts derived from Rosa-26mice, transgenic for the neomycin resistance gene. Selection was appliedfrom 48 hrs onwards by the addition of 400 μg/ml of G418.

A single colony of ESF116 was obtained expressing high levels of GFP(FIG. 10 a) which could easily be distinguished above the backgroundautofluorescence of the fibroblast feeder cells (FIG. 10 b). Thesetransfected ES cells were used to generate embryoid bodies using ourconventional protocol, with the exception that 400 μg/ml of G418 wereadded throughout the culture period. Embryoid bodies were screened usingconfocal fluorescence microscopy at various stages during theirdevelopment and were found to retain expression of GFP throughout (FIG.10 c-d). After plating onto tissue culture plastic in the presence ofGM-CSF and IL-3, dendritic cells were found to develop which continuedto express the EGFP transgene introduced into the parent ES cell line(FIG. 10 e-f). These results demonstrate the feasibility of introducingheterologous genes into DC for use in immunotherapy.

Example 6 Transfection of ESF116 with GFP Under the Control of the CD11cPromoter

Since upregulation of CD11c occurs during the maturation of DC, theexpression of EGFP under the control of the CD11c promoter will enablethe conditions for their maturation to be investigated. In order toprepare a suitable construct, the vector pBSCD11cβglob was obtained.This vector consists of an expression cassette composed of a 5.3 kbgenomic fragment containing the murine CD11c promoter and a rabbit βglobin fragment of approximately 1.2 kb providing an intron, all ofwhich is cloned into the pBSbluescript vector (Stratagene) [Kouskoff etal., 1993, Brocker et al., 1997]. The resulting construct contains aunique EcoRI cloning site and a polyadenylation signal.

A ˜1.8 kb PGKneopA cassette, consisting of the murine phosphoglyceratekinase-1 promoter and polyadenylation signals driving expression of aneomycin resistance gene, was cloned into the XhoI blunt site ofpBSCD11cβglob to generate pBSCD11cβglob (neo). This cassette providesG418 resistance for selection of stably transformed cells. The cassettewas inserted downstream of the CD11cβglob cassette to avoid anypossibility of interference with the DC specificity of the CD11cpromoter elements, and was cloned in the same orientation as theCD11cβglob cassette to avoid interference between the two and ensurethat any run-through from the CD11cβglob cassette will result only inincreased neomycin resistance.

EGFP was subcloned from pEGFP-N1 as a BglII/NotI blunt fragment to theEcORI blunt site of the CD11cβglob cassette in pBSCD11cβglob (neo). Themajority of the pEGFP-N1 multiple cloning site was retained tofacilitate the future generation of heterologous proteins fused to theN-terminus of EGFP under the control of the CD11cβglob expressioncassette. XhoI and EcoRI are suitable cloning sites for the generationof such fusion constructs. The vector may be linearized using NotI orXbaI, other potential linearization sites being ScaI and XmnI in the pBSvector sequence.

Example 7 Transfection of ESF116 with Fas-Ligand Gene

Constructs were prepared for the introduction of the Fas-ligand gene(accession number: U58995) into ESF116 so as to generate DCconstitutively expressing the protein. When administered to mice acrossan MHC barrier, these cells can be expected to attract naive T cellsspecific for the alloantigens they present: on activation, however, suchcells will be targeted for apoptosis by ligation of cell surface Fas.This strategy may deplete an animal of alloreactive T cells allowing thesubsequent acceptance of an organ allograft in spite ofhistoincompatibility.

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1. A first in vitro cell population comprising human embryonic stemcells and a second in vitro cell population comprising progeny of aportion of the first in vitro cell population, wherein the progeny aredendritic cells.
 2. The first and second cell populations of claim 1,wherein the dendritic cells are immature dendritic cells.
 3. The firstand second cell populations of claim 1, wherein the dendritic cells aremature dendritic cells.
 4. The first and second cell populations ofclaim 1, wherein the dendritic cells express B7-1.
 5. The first andsecond cell populations of claim 1, wherein the dendritic cells expressB7-2.
 6. The first and second cell populations of claim 1, wherein thedendritic cells express MHC II.
 7. The first and second cell populationsof claim 1, wherein the dendritic cells express CD11c.
 8. The first andsecond cell populations of claim 1, wherein the dendritic cells comprisea plurality of dendrite processes.
 9. The first and second cellpopulations of claim 1, wherein the human embryonic stem cells aregenetically modified.
 10. The first and second cell populations of claim1, wherein the dendritic cells are genetically modified.
 11. The firstand second cell populations of claim 1, wherein the dendritic cellsexpress IL-12.
 12. The first and second cell populations of claim 1,wherein the dendritic cells express IL-18.
 13. The first and second cellpopulations of claim 1, wherein the dendritic cells stimulate T cellproliferation.
 14. The first and second cell populations of claim 1,wherein the dendritic cells stimulate IL-2 expression in T cells. 15.The first and second cell populations of claim 1, wherein the humanembryonic stem cells form an embryoid body.
 16. The first and secondcell populations of claim 15, wherein the embryoid body is contained ina media comprising GM-CSF.
 17. The first and second cell populations ofclaim 1, wherein the embryonic stem cells are transfected with a nucleicacid.
 18. The first and second cell populations of claim 1, wherein thedendritic cells are transfected with a nucleic acid.